This is a competing renewal of an R01 (AI47062) initially submitted in response to the RFA: "Laboratory Methods to Assess Responses to HIV Vaccine Candidates." Since 2/1/00, substantial progress has been made on the first two Specific Aims of the original proposal, focused on development, optimization and utilization of a manual cytokine flow cytometry (CFC) assay for the detection of antigen-induced IFN gamma responses by CD4+ and CD8+ human T cells specific for the human immunodeficiency virus, type 1 (HIV-1). In studies that are currently ongoing, we anticipate that additional progress will be made in the use of this "Gag-IFN gamma, CFC assay" to determine whether various HIV-1 vaccines may induce specific CD4+ and/or CD8+ T cell immunity against HIV-1 in vivo. This work has generated 7 papers published in peer-reviewed journals and 5 abstracts presented at international meetings. Our objective now is to assess laboratory correlates of immune protection by automating new flow cytometric assays of antigen-specific immune responses, to identify candidate laboratory correlates of immune protection by comparing the results of these assays with results from other assays of immune phenotype and function in long-term non-progressors (LTNP), untreated subjects with progressive HIV-1 disease, and recipients of candidate HIV-1 vaccines, and to examine HIV-1-specific immune responses in HIV-1-infected individuals who appear to exhibit significant immune protection from HIV-1 disease. Specific Aim 1: To develop, optimize, and standardize the methods, hardware, and software for automated stimulus-response flow cytometric assays of immune function. The manual Gag-IFN gamma, CFC assay will be configured into an automated assay platform, amenable for use with additional stimulants and for the measurement of additional intracellular and cell surface markers. Specific Aim 2: To cross-sectionally compare Gag-IFN gamma CFC results to those obtained from other assays of immune phenotype and function in a) long-term non-progressors, b) untreated subjects with progressive HIV-1 disease, and c) recipients of candidate HIV-1 vaccine(s). The automated Gag-IFN gamma CFC assay will be compared to CFC assays, which measure additional intracellular and cell surface markers; and to more traditional measures of CD4+ and CD8+ T cell function (e.g., proliferation and cytolysis). Such comparisons will be made by cross-sectional analysis of two distinct groups of HIV-1-infected patients (long-term non-progressors and untreated subjects with progressive HIV-1 disease) and two distinct groups of HIV vaccinees ("responders" and "non-responders" as tested by lymphoproliferation, 51Cr release, and ELISPOT). Specific Aim 3 To prospectively characterize antigen-specific immunity in HIV-1-infected individuals who appear to exhibit significant immune protection from HIV-1 disease. Using a cohort of patients with partial virologic control, a prospective analysis will be carried out to define the temporal relationship between loss of immune function, as measured in CFC assay(s) developed in the first two Aims, and disease progression.